Protein-protein interactions are central to the process of signal transduction in biological systems. Transmembrane receptor tyrosine kinase (RTKs) play important roles in many aspects of cell growth and behavior. Activated RTKs, including one of the well-studied PDGF-beta receptor, have been shown to bind more than a dozen signal transduction molecules that contain Sre homology 2 (SH2) domains. These interactions depend on the recognition of phosphotyrosine residues in specific sequence contexts by the various SH2 domains. In the proposed research, selected recombinant SH2 domain constructs will be expressed in E. coli and purified by affinity chromatography. Test protein arrays on derivatized glass slides will be optimized with an SH2 domain from PI3-kinase based on its binding to a synthetic phosphopeptide. Additional recombinant SH2 domains will be arrayed and allowed to interact with extracts of cells expressing V-sis with a kinase dead or wild type PDGF receptor. We predict that this method will allow a rapid readout of which specific tyrosines are phosphorylated on the PDG receptor. These studies will aim to optimize array and binding parameters and thus to develop a new strategy for high throughput detection of phosphorylation dependent protein: protein interactions. PROPOSED COMMERCIAL APPLICATION: Commercial SH2 domain arrays will be developed to aid high throughput screening of novel interactions involved in cellular signal transduction and or possible novel kinases. Contracting in projects for high throughput screening of SH2 protein transactions.